J. Nat. Prod.

2004, 67, 1919-1924 1919

Chemical Constituents from the Colombian Medicinal Plant Maytenus laevis

Hiroyuki Nakagawa,† Yoshihisa Takaishi,*,† Yoshinori Fujimoto,‡ Carmenza Duque,§ Cristina Garzon,⊥
Mitsunobu Sato,# Masato Okamoto,# Tetsuya Oshikawa,# and Sharif Uddin Ahmed#
Faculty of Pharmaceutical Sciences, University of Tokushima, Sho-machi, Tokushima 770-8505, Japan, Department of
Chemistry and Materials Science, Tokyo Institute of Technology, Meguro, Tokyo 152-8551, Japan, Depatmento de Quimica,
and Institito de Ciencias Naturales, Universidad Nacional de Colombia, AA 14490, Bogota, Colombia, and Schoool of
Dentistry, University of Tokushima, Kuramoto-cho, Tokushima 770-8504, Japan

Received January 13, 2004

The methanol extract of the bark of the Colombian medicinal plant Maytenus laevis gave six new
compounds and 28 known compounds. The structures of the new and known compounds were elucidated
on the basis of spectroscopic evidence. Several of these compounds were screened for cytokine-inducing
activity on human PBMCs to investigate antitumor effects, and canophyllol (12) demonstrated the most
effective induction of the cytokines.

Species belonging to the genus Maytenus (Celastraceae)
have been used as a traditional medicine in the Amazonian
region against cancer, rheumatism, and inflammation.1-3
The bark of Maytenus laevis, known by a vernacular name
meaning shaking waist, “chuchuhuasha” or “chuchuasi”,
is used as an alcoholic infusion, generally in “aguardiente”,
for the treatment of rheumatism and as a tonic and an
aphrodisiac.4-6 Antitumor, antiinflammatory, and radio-
protective activities have been reported from M. laevis by
Gonzalez et al.2 In the present study, we report the
isolation and structural elucidation of six new and 28
known compounds from the methanol extract of the bark
of M. laevis. The cytokine-inducing activity of some of the
isolated compounds is also reported.
Repeated column chromatography of the ethyl acetate-
soluble fraction from the methanol extract of the bark of
M. laevis yielded six new compounds, including one ses-
quiterpene pyridine alkaloid, one flavonoid, two triter-
penoids, one lignan, one iridoid, and 28 known compounds.
Compound 1 was assigned the molecular formula
C41H47O17N on the basis of HRFABMS. The IR spectrum
revealed hydroxy and ester carbonyl bands (3457 and 1744
cm-1). The 1H NMR spectrum showed four acetyl groups
(δH 2.33, 2.22, 2.12, and 1.42), one benzoyl group [δH 7.96
(2H, d, J ) 7.4 Hz), 7.57 (1H, t, J ) 7.4 Hz), and 7.43 (2H,
t, J ) 7.7 Hz)], two tertiary methyl groups (δH 1.72, 1.64),
two sets of methylene protons [δH 6.00, 3.71 (each 1H, d, J
) 11.4 Hz), 5.48, 4.81 (each 1H, d, J ) 13.6 Hz)], and seven
methine protons [δH 7.07, 5.76, 5.54, 5.41, 4.84, 4.14, and
2.36]. It also showed one evonic acid moiety, 2,3-disubsti-
tuted pyridine (δH 8.77, 8.14, and 7.34), two secondary
methyl groups [δH 1.44 and 1.20 (each 3H, d, J ) 6.9 Hz)],
and two methine protons [δH 4.72 and 2.59 (each 1H, q, J
) 6.9 Hz)]. The 13C NMR spectra of 1 indicated the
presence of eight methyl carbons, two methylene carbons
attached to an oxygen function, six methine carbons
attached to an oxygen function, one methine carbon, four
ester carbonyl carbons, four quaternary carbons, one ben-
zoyl group [δC 165.0 (s), 133.8 (d), 129.9 (d) × 2, 129.3 (s),
* To whom correspondence should be addressed. Tel: 81-886337275.
Fax: 81-886339501. E-mail: takaishi@ph.tokushima-u.ac.jp.
†
Faculty of Pharmaceutical Sciences, University of Tokushima.
‡
§
Tokyo Institute of Technology.
Depatmento de Quimica, Universidad Nacional de Colombia.
⊥
Institito de Ciencias Naturales, Universidad Nacional de Colombia.
#
Schoool of Dentistry, University of Tokushima.
10.1021/np040006s CCC: $27.50 © 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/28/2004
and 128.8 (d) × 2], and one evonic acid moiety [δC 174.4
(s), 168.2 (s), 165.0 (s), 150.9 (d), 138.8 (d), 126.0 (s), 121.7
(d), 45.2 (d), 36.6 (d), 12.1 (q), and 9.8 (q)]. Owing to the
presence of ester carbons and a pyridine ring moiety, 1 was
assumed to be a sesquiterpene pyridine alkaloid derived
from dihydroagarofuran polyol esters found in the Celastra-
ceae.7-9 The NMR spectra of 1 suggested that it was an
evonine-type sesquiterpene alkaloid,7-13 possessing four
acetyl groups and one benzoyl group. The 1H-1H COSY
and the coupling pattern among the seven methine protons
[δH 5.76 (H-1), 4.14 (H-2), 4.84 (H-3), 7.07 (H-5), 2.36 (H-
6), 5.54 (H-7), and 5.41 (H-8)] revealed their connections
in the dihydroagarofuran core. In the HMBC spectrum of
1, the proton signal at δH 5.76 (H-1) was correlated with
the carbon signals at δC 53.0 (C-9) and 60.5 (C-11); the
proton signal at δH 1.64 (H-12) with the carbon signals at
δC 78.3 (C-3), 70.7 (C-4), and 94.8 (C-10); the proton signal
at δH 7.07 (H-5) with the carbon signal at δC 94.8 (C-10);
the proton signal at δH 5.41 (H-8) with the carbon signal
at δC 53.0 (C-9); the proton signal at δH 1.72 (H-14) with
the carbon signals at δC 50.6 (C-6), 84.3 (C-13), and 70.5
(C-15). The proton signal at δH 4.84 (H-3) was correlated
with the carbon signal at δC 174.4 (C-11′); the proton signal
at δH 2.59 (H-8′) with the carbon signals at δC 36.6 (C-7′)
and 174.4 (C-11′); the proton signal at δH 8.14 (H-4′) with
the carbon signal at δC 168.2 (C-12′); the proton signal at
δH 6.00 (H-15) with the carbon signal at δC 168.2 (C-12′)
in the HMBC spectrum; these facts clearly indicated that
the macrocyclic structure was formed by ester linkages
between the dihydroagarofuran core and evonic acid at
positions C-3 and 15.
On comparing the 13C NMR spectroscopic data of 1 with
the literature, compound 1 was found to be similar to
hyponine C,14 except for the presence of five acetyl groups
in hyponine C compared to four in 1. From the HMBC
spectrum of 1, the locations of the ester groups were
determined as follows: the proton signals at δH 7.07 (H-5)
and 5.41 (H-8) showed long-range correlations with the
carbonyl carbons of the acetyl groups at δC 170.0 and 169.2,
respectively. The 1H chemical shifts of H-1 (δH 5.76), H-2
(δH 4.14), and H-7 (δH 5.54) revealed acylation at C-1 and
C-7. Thus, the four acetyl groups were assigned at positions
C-1, -5, -7, and -8. The benzoyl group was assigned at
position C-11 from an NOE correlation between the proton
at δH 7.96 (ortho-Bz) and the proton at δH 4.81 (H-11). In
1920 Journal of Natural Products, 2004, Vol. 67, No. 11
the NOESY spectrum, the proton at δH 7.07 (H-5) was
correlated with the protons at δH 5.48 (H-11), 2.36 (H-6),
and 1.64 (H-12); the proton at δH 5.41 (H-8) with the
protons at δH 5.76 (H-1) and 5.54 (H-7); the proton at δH
5.76 (H-1) with the protons at δH 5.41 (H-8) and 4.14 (H-
2). The proton at δH 2.36 (H-6) was correlated with the
proton at δH 3.71 (H-15). This evidence proved that the
relative configuration of the ester groups was 1β, 2β, 5R,
Notes
7β, and 8β, and that of the two methyl groups was 12β,
14R. Therefore, the structure of 1 was formulated as
7-(acetyloxy)-O11-benzoyl-O2,11-deacetyl-7-deoxoevonine, as
shown.
Compound 2 was assigned the molecular formula
C16H14O8 on the basis of HRFABMS. The 1H NMR spec-
trum showed four aromatic protons [δH 6.56 (2H, brs), 5.97
and 5.92 (each 1H, brs)], two methine protons [δH 5.26 and
4.20 (each 1H, d, J ) 2.1 Hz)], and one methoxyl group
(δH 3.80). The 13C NMR spectra of 2 indicated the presence
of two methine carbons attached to an oxygen function (δC
85.4 and 75.8), 12 aromatic carbons δC 171.3 (s), 168.6 (s),
167.0 (s), 154.3 × 2 (s), 139.4 (s), 135.9 (s), 110.4 × 2 (d),
104.6 (s), 99.9 (d), and 99.0 (d)], one carbonyl carbon (δC
199.0), and one methoxyl group (δC 63.5). The above
spectroscopic data suggested that 2 was a flavonoid and
were very similar to those of 4′-O-methyl-2,3-dihydromyrice-
tin ()pallasiin).15 However, the 1H NMR spectrum of 2 was
considerably different from that of pallasiin in regard to
the coupling constant of H-2 and Η-3. The value of the
coupling constant in 2 was J2,3 ) 2.1 Hz versus J2,3 ) 11.0
Hz in pallasiin. From this difference, 2 was suggested not
to be a 2,3-trans-3-hydroxyflavanone as pallasiin, but to
be a 2,3-cis isomer. The absolute configurations at C-2 and
C-3 were deduced from the circular dichroism (CD) spec-
trum of 2. The CD spectrum of 2 showed negative and
positive Cotton effects at 292 and 339 nm, respectively. The
CD curves and maxima of 2 showed the characteristic
pattern of a 2R,3S-dihydroflavanol.16-19 Therefore, 2 was
determined as (2R,3S)-4′-O-methyl-2,3-dihydromyricetin.
Compound 3 was assigned the molecular formula
C30H48O4 on the basis of HRFABMS. The 1H NMR spec-
trum showed seven tertiary methyl groups (δH 1.80, 1.26,
1.22, 1.15, 1.07, 1.00, and 0.91), one olefinic proton [δH 5.41
(1H, brs)], and two methine protons [δH 4.00 (1H, brdd, J
) 4.5, 2.7 Hz), 3.61 (1H, brs)] attached to an oxygen
function. The 13C NMR spectra of 3 indicated the presence
of seven methyl carbons, nine methylene carbons, and two
methine carbons (δC 75.3 and 75.2) attached to an oxygen
function, three methine carbons, seven quaternary carbons,
and one carboxylic acid (δC 181.4). This suggested that 3
was a triterpenoid derivative. Many types of triterpenoids
have been isolated from Celastraceae plants;20-22 compound
3 was assumed to be an oleanane-type triterpenoid from
the carbon number (C30) and the presence of seven tertiary
methyl groups, one olefinic methine, and one carboxylic
acid.
A detailed analysis of the 1H-1H COSY and HMBC
spectral data as well as the 1H and 13C NMR data led to
the conclusion that the structure of 3 was 3,22-dihydroxy-
olean-12-en-29-oic acid. The 13C NMR data of 3 were
similar to those of 3β,22β-dihydroxyolean-12-en-29-oic acid
(22) except for C-3.23 These data suggested that the A-E
ring systems were the same as in 22. The configuration of
hydroxy groups on C-3 and C-22 were determined as
follows. The signal at δH 3.61 (brs) suggested an R-config-
uration for the hydroxy group at C-3, because its coupling
constant was small and the C-24 carbon signal of 3
suggested a downfield shift (δC 22.8) in comparison with
that of 22 (δC 16.6). The 22-hydroxy group was deduced to
be in a β-configuration from the NOESY spectrum; the
proton at δH 1.26 (H-28) correlated with the protons at δH
2.08 (H-21β) and 2.46 (H-18); the proton at δH 4.00 (H-22)
with the proton at δH 2.64 (H-21R); the proton at δH 1.80
(H-30) with the proton at δH 2.46 (H-18). Ring E is thus in
a chair conformation that placed the C-20R carboxylic acid
and the C-22β hydroxy group in an equatorial and axial
Notes
orientation, respectively. From the above evidence, 3 was
shown to be 3R,22β-dihydroxyolean-12-en-29-oic acid. Four
stereoisomeric forms of 3,22-dihydroxyolean-12-en-29-oic
acid are possible with differing configurations at C-3 and
C-22. We also isolated the remaining three isomers (20,
22, and 24). Although those isomers have been individually
isolated from several plants,23-25 it is interesting that this
was the first time that all isomers were isolated from one
plant.
Compound 4 was assigned the molecular formula
C39H56O5 on the basis of HRFABMS. The 1H NMR spec-
trum showed seven methyl groups [δH 1.09 (3H, s), 0.98
(6H, s), 0.92 (3H, s), 0.91 (3H, d, J ) 5.9 Hz), 0.89 (3H, s),
and 0.79 (3H, d, J ) 5.8 Hz)], three olefinic protons [δH
7.51, 6.21 (each 1H, d, J ) 15.9 Hz), and 5.11 (1H, brs)],
one set of methylene protons [δH 3.52 and 3.14 (each 1H,
d, J ) 11.0 Hz)], six methine protons [δH 4.59 (1H, t, J )
8.9 Hz), 1.55, 1.37, 1.36, 0.87, and 0.82 (each 1H, m)], and
three aromatic protons [δH 7.03 (1H, d, J ) 1.8 Hz), 6.93
(1H, dd, J ) 8.2, 1.8 Hz), and 6.80 (1H, d, J ) 8.2 Hz)].
The coupling pattern of the aromatic protons showed the
presence of a 1,3,4-substituted ring. In addition, the
coupling constant of the protons at δH 7.51 and 6.21 was
15.9 Hz, indicating the presence of two trans-olefinic
protons. From these results, compound 4 was judged to be
the caffeoyl ester of a triterpene.
The 13C NMR spectral data of 4 were very similar to
those of uvaol26 except for the chemical shift of C-3 and
the O-caffeoyl group. In the HMBC spectrum, the proton
at δH 4.59 (H-3) correlated with the carbons at δC 17.0 (C-
24) and 167.9 (C-9′), indicating that the O-caffeoyl group
was located at C-3. Owing to the correlation of H-3 and
H-5 present in the NOESY spectrum, the relative config-
uration of the O-caffeoyl group was indicated to be 3β.
Therefore, the structure of 4 was determined as 28-
hydroxy-12-ursene-3β-yl-caffeate ()uvaol-3β-yl-caffeate).
Compound 5 was assigned the molecular formula
C35H44O14 on the basis of HRFABMS. The 1H and 13C NMR
spectra showed four methoxyl groups, four sets of meth-
ylenes, two methine protons, nine aromatic protons, and
one sugar moiety. The 1H and 13C NMR spectra, analyzed
by 2D NMR, suggested the presence of one secoisolaricir-
esinol,27 one β-glucose, one 1,3,4-trisubstituted benzoyl
group, and two methoxyl groups. The benzoyl group was
defined as veratroyl on the basis of the HMBC spectrum.
Moreover, the correlation between the anomeric proton (δH
4.83, H-1′′) and H-5 (δH 6.89) in the NOESY spectrum and
the downfield shifts of C-1′′ (δC 102.8) and C-6′′ (δC 65.2)
of the β-glucose moiety, when compared with those (δC 96.8
and 61.8) of β-D-glucose,28 suggested that the glucose
moiety and the veratroyl group were located at C-4 and
C-6′′, respectively. A comparison of the NMR data of 5 with
(-)-secoisolariciresinol 4-O-β-D-(6-O-vanilloyl)glucopyrano-
side29 showed almost the same chemical shift values, except
for one methoxy group. Thus, 5, [R]D -31.2°, was identified
as (-)-secoisolariciresinol 4-O-β-D-(6-O-veratroyl)glucopy-
ranoside.
Compound 6 was assigned the molecular formula
C25H34O13 on the basis of HRFABMS. The 1H NMR
spectrum showed three methoxyl groups [δH 3.87 (6H, s),
3.82 (3H, s)], one set of methylene protons [δH 2.28 (1H,
dd, J ) 14.3, 6.2 Hz), 2.06 (1H, dd, J ) 14.3, 3.4 Hz)], two
olefinic protons [δH 6.24 (1H, dd, J ) 6.3, 2.1 Hz), 4.99 (1H,
dd, J ) 6.3, 2.7 Hz)], two methine protons [δH 5.51 (1H, d,
J ) 2.5 Hz), 5.06 (1H, m)] attached to oxygen functions,
two methine protons [δH 3.01 (1H, m), 2.62 (1H, brd, J )
9.0 Hz)], two aromatic methine protons [δH 7.36 (2H, brs)],
Journal of Natural Products, 2004, Vol. 67, No. 11 1921
and one sugar moiety [δH 4.66 (1H, d, J ) 7.9 Hz), 3.88
(1H, m), 3.65 (1H, dd, J ) 11.8, 5.8 Hz), 3.36 (1H, t, J )
8.7 Hz), 3.34 (1H, m), 3.26 (1H, m), and 3.18 (1H, t, J )
8.7 Hz)]. The 13C NMR spectra of 6 indicated the presence
of one carboxylic carbon (δC 167.4), three methoxyl carbons
(δC 61.2, 56.8 × 2), one methylene carbon, two olefinic
carbons (δC 141.3, 104.4), two methine carbons (δC 93.5,
81.2) attached to an oxygen function, two methine carbons,
six aromatic carbons [δC 154.4 × 2, 143.7, 126.9 (each s),
and 108.2 × 2 (d)], and one sugar moiety [δC 99.4, 78.3,
78.0, 74.8, 71.7 (each d), and 62.9 (t)]. A detailed analysis
of the 1H-1H COSY, HSQC, and HMBC spectra suggested
that 6 was an ajugol-type iridoid glycoside esterified with
a 3,4,5-trimethoxybenzoyl group. The 1H and 13C NMR data
of 6 were very similar to those of ajugol30 except for the
3,4,5-trimethoxybenzoyl moiety. In the 13C NMR spectrum,
the carbon signal at δC 81.2 (C-6) shifted downfield (∆ +3.0
ppm) and the carbon signals at δC 39.6 (C-5) and 47.7 (C-
7) shifted upfield (∆ -1.7 and -2.3 ppm, respectively) in
comparison with those of ajugol. These facts indicated that
the 3,4,5-trimethoxybenzoyl group was attached at C-6.
Thus, the structure of 6 was determined as 6-O-(3′,4′,5′-
trimethoxybenzoyl)ajugol.
Known compounds were also isolated; ebenifoline E-II
(7),31 mayteine (8),9 p-hydroxybenzoic acid (9),32 ebenifoline
E-III (10),33 lambertic acid (11),34 canophyllol (12),25
3-oxoolean-12-en-oic acid (13),35 β-sitosterol (14),36 wilfor-
lide B (15),37 abruslactone A (16),25 3-epiabruslactone A
(17),38 salaspermic acid (18),39 22β-hydroxy-3-oxoolean-12-
en-oic acid (19),23 maytenfolic acid (20),25 mearnsetin (21),15
3β,22β-dihydroxyolean-12-en-29-oic acid (22),23 triptocallic
acid A (23),40 triptocallic acid D (24),24 (-)-4′-O-methylepi-
gallocatechin (25),41 3′,4′-di-O-methyl-(-)-epicatechin (26),42
epikatonic acid (27),43 6-O-3′,4′-dimethoxybenzoyl ajugol
(28),44 isoverbascoside (29),45 ent-isolariciresinol (30),46 6-O-
p-hydroxybenzoyl ajugol (31),44 6-O-4′′-hydroxy-3′′-meth-
oxybenzoyl ajugol (32),47 ourateaproanthocyanidin A (33),48
and ajugol (34)30 were identified from spectral data com-
parison with the literature, respectively.
Furthermore, several of the isolated compounds were
assayed for cytokine-inducing activity on human peripheral
blood mononuclear cells (PBMCs) to investigate antitumor
effects. OK-432 used as a positive control induced all of
the cytokines examined except for interleukin (IL)-4 (Table
1). It has been reported that OK-432 elicits antitumor
effects by stimulating immunocompetent cells, such as
macrophages, T cells, and natural killer cells and induces
multiple cytokines including IL-1, IL-2, IL-6, tumor necro-
sis factor (TNF)-R, and interferon (IFN)-γ.49-53 One or 10
µg/mL of the compounds was added into the PBMC culture.
Forty-eight hours later, cytokines in the supernatants were
measured. Data are shown in Table 1. All of the cytokines
examined were not detected in a supernatant derived from
untreated PBMC culture. IL-6, one of the helper T lym-
phocytes 2 (Th2)-type cytokines, was induced by the
treatment with most of the compounds tested, especially
compounds 8, 12, and 15 (>1000 pg/mL). IL-12 and TNF-
R, Th1-type cytokines, were secreted by the PBMCs stimu-
lated with several compounds, while compound 12 was
most effective in the induction of the cytokines. None of
the compounds induced IFN-γ, IL-4, or IL-10 in the current
in vitro system (data not shown).
Experimental Section
General Experimental Procedures. NMR spectra (400
MHz for 1H NMR, 100 MHz for 13C NMR, both using TMS as
internal standard) were measured on a Bruker ARX-400
instrument and a Bruker AVANCE-400 instrument. MS were
1922 Journal of Natural Products, 2004, Vol. 67, No. 11 Notes

Table 1. Cytokine Induction by the Compounds on Human (CHCl3), GPC (MeOH), Si HPLC (CHCl3-MeOH), and pre-
PBMCsa parative TLC (CHCl3-MeOH) to give 27 (5.9 mg). Fraction
secreted cytokines 3.4 was subjected to Si HPLC (CHCl3-MeOH) and Si HPLC
(n-hexanes-EtOAc) to give 4 (7.4 mg). Fraction 4 (4.5 g) was
IL-6 (pg/mL) IL-12 (pg/mL) TNF-R (pg/mL) chromatographed on a Toyopearl HW-40 column to give 10
compd 1 µg/mL 10 µg/mL 1 µg/mL 10 µg/mL 1 µg/mL 10 µg/mL fractions (4.1-4.10). Fraction 4.2 was separated by Si HPLC
(n-hexane-EtOAc) to give 7 (18 mg), 8 (163 mg), and seven
1 136.1 110.1 N.D. N.D. N.D. N.D.
2 279.4 470.4 N.D. N.D. N.D. N.D. other fractions (4.2.1-4.2.7). Fraction 4.2.6 was separated by
3 32.2 16.2 N.D. N.D. N.D. N.D. preparative TLC (CHCl3-MeOH) to give 1 (4.5 mg) and 10
4 28.5 607.1 N.D. N.D. N.D. 27.9 (13 mg). Fraction 4.3 (yield 0.6 g of 2.4 g) was subjected to
7 463.0 260.8 17.3 N.D. 26.7 N.D. ODS (MeOH-H2O) to give 15 (8.8 mg), 16 (7.0 mg), 17 (8.0
8 1245.2 1576.2 14.3 14.7 22.0 24.6 mg), 18 (6.5 mg), and 17 other fractions (4.3.1-4.3.16).
10 N.D. N.D. N.D. N.D. N.D. N.D. Fraction 4.3.10 was further separated by Si HPLC (CHCl3-
11 59.8 1454.4 N.D. N.D. N.D. N.D. MeOH) to give 19 (3.5 mg), 20 (18 mg), and nine other fractions
12 2579.3 2579.2 47.3 104.3 132.5 322.9 (4.3.10.1-4.3.10.9). Fractions 4.3.10.5 and 4.3.10.6 were sepa-
15 1477.6 451.1 N.D. N.D. N.D. N.D.
rated by GPC (MeOH) to give 22 (8.0 mg) and 23 (4.9 mg),
16 22.5 777.2 N.D. 28.3 N.D. 22.7
17 N.D. 308.4 N.D. N.D. N.D. N.D. respectively. Fraction 4.3.13 was subjected to Si HPLC (CHCl3-
18 857.3 109.6 21.6 N.D. 54.5 N.D. MeOH) and GPC (MeOH) to give 24 (7.6 mg). Fraction 4.3.14
20 380.0 76.1 N.D. N.D. N.D. N.D. was separated by GPC (MeOH) to give 3 (6.7 mg). Combined
21 783.6 203.4 14.3 N.D. 31.3 N.D. fractions 4.6 and 4.7 were separated by Si HPLC (hexane-
22 57.5 297.6 N.D. N.D. N.D. N.D. EtOAc) to give 9 (28 mg). Fraction 4.10 was subjected to Si
23 705.7 10.6 N.D. N.D. N.D. N.D. HPLC (n-hexane-EtOAc) and preparative TLC (CHCl3-
24 793.2 N.D. N.D. N.D. N.D. N.D. MeOH) to give 2 (14 mg) and 21 (6.1 mg). Fraction 5 (1.5 g)
25 N.D. 662.9 N.D. N.D. N.D. N.D. was subjected to Toyopearl HW-40, GPC (MeOH), and ODS
26 473.4 198.6 N.D. N.D. N.D. N.D.
(MeOH-H2O) to give 25 (36 mg) and 26 (5.6 mg). Fraction 7
27 16.2 22.9 N.D. N.D. N.D. N.D.
28 849.5 38.1 27.2 N.D. 12.6 N.D.
(yield 3.0 g of 9.2 g) was chromatographed on a Sephadex LH-
29 101.5 175.5 N.D. N.D. N.D. N.D. 20 (MeOH) column to give 10 fractions (7.1-7.10). Fraction
32 N.D. N.D. N.D. N.D. N.D. N.D. 7.3 was separated by ODS (MeOH-H2O) to give 30 (2.7 mg).
33 28.1 174.4 N.D. N.D. N.D. N.D. Fraction 7.7 was subjected to ODS (MeOH-H2O) and GPC
OK-432 1824.5 2555.2 462.7 542.3 315.8 278.4 (MeOH) to give 33 (11 mg). Fraction 10 (yield 10 g of 19 g)
a Human PBMCs (1 × 106 /mL) were stimulated with the was chromatographed on a Diaion HP-20 [H2O-MeOH (1:0,
compounds (1 or 10 µg/mL) for 48 h at 37 °C, then cytokines in
9:1, 8:2, 7:3, 1:1, MeOH)] column to give 13 fractions (10.1-
the supernatants of these cultures were analyzed by ELISA. Data 10.13). Fraction 10.6 was subjected to GPC (MeOH), ODS
represent mean value of triplicate samples. N.D. ) not detected (MeOH-H2O), and preparative TLC (CHCl3-MeOH-H2O) to
(<7.8 pg/mL). give 34 (0.9 mg). Combined fractions 10.8 and 10.9 were
separated by LH-20 (MeOH) and GPC (MeOH) to give 29 (7.9
obtained on a JEOL JMSD-300 instrument. CC: silica gel 60 mg), 31 (4.2 mg), and 32 (8.3 mg). Fraction 10.10 was subjected
(Merck), Toyopearl HW-40 (Tosoh), Sephadex LH-20 (Phar- to LH-20 (MeOH), GPC (MeOH), and ODS (MeOH-H2O) to
give 5 (2.9 mg), 6 (3.8 mg), and 28 (19 mg).
macia), and Diaion HP-20 (Nippon Rensui); HPLC: GPC (gel-
Treatment of Human PBMCs and Cytokine-Inducing
permeation chromatography: Shodex H-2001, 2002, CHCl3;
Assay. PBMCs were isolated from heparinized venous blood
Asahipak, GS-310 2G, MeOH), silica gel HPLC (Si1: YMC-
by Ficoll-Hypaque gradient density centrifugation according
Park SIL-06 SH-043-5-06, 250 × 20 mm, hexane-EtOAc to standard procedures.54 PBMCs (1 × 106/mL) were cultured
system; Si2: Hibar RT 250-25, LiChrosorb Si 60, CHCl3- in RPMI 1640 medium containing 10% fetal calf serum in the
MeOH system); ODS: Hibar RT 250-25, LiChrosorb RP 18. presence or absence of the compounds (1 or 10 µg/mL) for 48
IR spectra were recorded on a 1720 infrared Fourier transform h at 37 °C, then cytokines in the supernatants of these cultures
spectrometer (Perkin-Elmer), and the CD spectrum was were analyzed by commercial ELISA kits. OK-432 (Chugai
measured on a JASCO CD-J600 spectropolarimeter. Optical Pharmaceutical Co., Ltd., Tokyo, Japan), a Streptococcus
rotations were measured with a JASCO DIP-370 digital pyogenes-derived immunopotentiator that is commonly used
polarimeter. for immunotherapy in malignancies, was used as a positive
Plant Material. The bark of Maytenus laevis was collected control. ELISA kits for human IFN-γ, TNF-R, IL-4, IL-6, IL-
from Leticia in Colombia in August 2000 and identified by one 10, and IL-12 were purchased from BioSource International,
of the authors (C.G.). A voucher specimen (OOJC007) is Inc. (Camarillo, CA).
deposited in the Institute de Ciencias Naturales, Universidad Compound 1: white amorphous powder; [R]D +4.3° (c 0.2,
Nacional de Colombia, Colombia. CHCl3-MeOH); IR (KBr) νmax 3457, 2929, 1744, 1371, 1249,
Extraction and Isolation. The dried bark (1.9 kg) of M. 1118, 1057 cm-1; 1H NMR (CDCl3, 400 MHz) δ 8.77 (1H, brs,
laevis was extracted with MeOH. The MeOH extracts were H-6′), 8.14 (1H, brd, J ) 6.6 Hz, H-4′), 7.96 (2H, d, J ) 7.4
concentrated in vacuo to give a residue (248 g), which was Hz, ortho-Bz), 7.57 (1H, t, J ) 7.4 Hz, para-Bz), 7.43 (2H, d, J
suspended in H2O and partitioned sequentially with EtOAc ) 7.7 Hz, meta-Bz), 7.34 (1H, brs, H-5′), 7.07 (1H, brs, H-5),
and n-BuOH. The EtOAc layer was concentrated to give a 6.00 (1H, d, J ) 11.4 Hz, H-15a), 5.76 (1H, d, J ) 3.8 Hz, H-1),
residue (113 g), which was subjected to silica gel column 5.54 (1H, t, J ) 5.5 Hz, H-7), 5.48 (1H, d, J ) 13.6 Hz, H-11a),
chromatography (1.2 kg, 11 × 100 cm). The column was eluted 5.41 (1H, d, J ) 5.8 Hz, H-8), 4.84 (1H, d, J ) 2.2 Hz, H-3),
with solvents of increasing polarity (n-hexane-EtOAc, EtOAc, 4.81 (1H, d, J ) 13.6 Hz, H-11b), 4.72 (1H, q, J ) 6.9 Hz, H-7′),
EtOAc-MeOH, MeOH) to give 12 major fractions (1-12). 4.14 (1H, brs, H-2), 3.71 (1H, d, J ) 11.4 Hz, H-15b), 2.59 (1H,
Fraction 2 (3.9 g) was chromatographed on a Toyopearl HW- q, J ) 6.9 Hz, H-8′), 2.36 (1H, d, J ) 3.8 Hz, H-6), 2.33 (3H, s,
40 column (CHCl3-MeOH, 2:1) to give four fractions (2.1- 1-Ac), 2.22 (3H, s, 5-Ac), 2.12 (3H, s, 7-Ac), 1.72 (3H, s, H-14),
2.4). Fraction 2.3 was separated by ODS (MeOH-H2O, 9:1) to 1.64 (3H, s, H-12), 1.44 (1H, d, J ) 6.9 Hz, H-9′), 1.42 (3H, s,
give 18 fractions (2.3.1-2.3.18). Combined fractions 2.3.1 and 8-Ac), 1.20 (1H, d, J ) 6.9 Hz, H-10′); 13C NMR (CDCl3, 100
2.3.2 crystallized from MeOH to give 14 (33 mg). Fraction MHz) δ 75.5 (C-1), 70.3 (C-2), 78.3 (C-3), 70.7 (C-4), 74.1 (C-
2.3.15 was separated by preparative TLC (CHCl3-MeOH, 95: 5), 50.6 (C-6), 69.1 (C-7), 71.8 (C-8), 53.0 (C-9), 94.8 (C-10),
5) to give 12 (11 mg) and 13 (3.5 mg). Fraction 2.4 was 60.5 (C-11), 23.2 (C-12), 84.3 (C-13), 18.6 (C-14), 70.5 (C-15),
subjected to Si HPLC (CHCl3-MeOH, 98:2) to give 11 (33 mg). 165.0 (C-2′), 126.0 (C-3′), 138.8 (C-4′), 121.7 (C-5′), 150.9 (C-
Fraction 3 (1.5 g) was chromatographed on a Toyopearl HW- 6′), 36.6 (C-7′), 45.2 (C-8′), 12.1 (C-9′), 9.8 (C-10′), 174.4 (C-
40 column and silica gel column (CHCl3-MeOH) to give five 11′), 168.2 (C-12′), 170.3, 21.6 (1-Ac), 170.0, 21.8 (5-Ac), 170.2,
fractions (3.1-3.5). Fraction 3.3 was separated by GPC 21.1 (7-Ac), 169.2, 20.1 (8-Ac), 165.0, 129.3, 129.9 × 2, 128.8
Notes
× 2, 133.8 (11-Bz); HRFABMS m/z 826.2911 [M + H]+ (calcd
for C41H48O17N, 826.2922).
Compound 2: yellow amorphous powder; [R]D -59.5° (c 0.7,
MeOH); IR (KBr) νmax 3492, 2938, 2359, 1639, 1594, 1462,
1369, 1166 cm-1; CD (c 0.0000898, MeOH) [θ] (nm) -3.4 ×
10-4 (292) (negative maximum), 0 (317), +1.6 × 10-4 (339)
(positive maximum); 1H NMR (CD3OD, 400 MHz) δ 6.56 (2H,
brs, H-2′, 6′), 5.97 (1H, brs, H-8), 5.92 (1H, brs, H-6), 5.26 (1H,
d, J ) 2.1 Hz, H-2), 4.20 (1H, d, J ) 2.1 Hz, H-3), 3.80 (3H,
brs, 4′-OMe); 13C NMR (CD3OD, 100 MHz) δ 85.4 (C-2), 75.8
(C-3), 199.0 (C-4), 168.6 (C-5), 99.9 (C-6), 171.3 (C-7), 99.0 (C-
8), 167.0 (C-9), 104.6 (C-10), 135.9 (C-1′), 110.4 × 2 (C-2′, 6′),
154.3 (C-3′, 5′), 139.4 (C-4′), 63.5 (4′-OMe); HRFABMS m/z
333.0580 [M - H]- (calcd for C16H13O8, 333.0610).
Compound 3: white amorphous powder; [R]D +44.4° (c 0.7,
CHCl3-MeOH); IR (KBr) νmax 3438, 2945, 1705 cm-1; 1H NMR
(C5D5N, 400 MHz) δ 5.41 (1H, brs, H-12), 4.00 (1H, brdd, J )
4.5, 2.7 Hz, H-22), 3.61 (1H, brs, H-3), 2.74 (1H, t, J ) 13.8
Hz, H-19a), 2.64 (1H, dd, J ) 13.7, 2.8 Hz, H-21a), 2.46 (1H,
brd, J ) 13.8 Hz, H-18), 2.08 (1H, m, H-21b), 2.03 (1H, m,
H-2a), 1.98 (2H, m, H-11), 1.97 (1H, m, H-16a), 1.87 (1H, m,
H-9), 1.84 (1H, m, H-15a), 1.80 (3H, s, H-30), 1.78 (2H, m,
H-1a, 2b), 1.68 (1H, m, H-19b), 1.67 (1H, m, H-5), 1.55 (1H,
m, H-7a), 1.52 (2H, m, H-6), 1.44 (1H, m, H-16b), 1.42 (1H, m,
H-1b), 1.33 (1H, m, H-7b), 1.26 (3H, s, H-28), 1.22 (3H, s, H-24),
1.15 (3H, s, H-27), 1.07 (3H, s, H-26), 1.01 (1H, m, H-15b),
1.00 (3H, s, H-25), 0.91 (3H, s, H-23); 13C NMR (C5D5N, 100
MHz) δ 33.8 (C-1), 26.4 (C-2), 75.2 (C-3), 37.9 (C- 4), 49.2 (C-
5), 18.6 (C-6), 33.2 (C-7), 40.2 (C-8), 47.8 (C-9), 37.4 (C-10),
23.8 (C-11), 123.3 (C-12), 144.2 (C-13), 42.5 (C-14), 26.3 (C-
15), 28.9 (C-16), 38.0 (C-17), 44.7 (C-18), 41.5 (C-19), 42.5 (C-
20), 37.7 (C-21), 75.3 (C-22), 29.2 (C-23), 22.8 (C-24), 15.7 (C-
25), 17.3 (C-26), 25.5 (C-27), 20.9 (C-28), 181.4 (C-29), 24.9 (C-
30); HRFABMS m/z 471.3519 [M - H]- (calcd for C30H47O4,
471.3474).
Compound 4: yellow amorphous powder; [R]D +21.5° (c 0.7,
MeOH); IR (KBr) νmax 3432, 2925, 1681, 1605, 1274, 1182 cm-1;
1
H NMR (CDCl3, 400 MHz) δ 7.51 (1H, d, J ) 15.9 Hz, H-7′),
7.03 (1H, d, J ) 1.8 Hz, H-2′), 6.93 (1H, dd, J ) 8.2, 1.8 Hz,
H-6′), 6.80 (1H, d, J ) 8.2 Hz, H-5′), 6.21 (1H, d, J ) 15.9 Hz,
H-8′), 5.11 (1H, brs, H-12), 4.59 (1H, t, J ) 8.9 Hz, H-3), 3.52,
3.14 (each 1H, d, J ) 11.0 Hz, H-28), 2.26 (2H, m, H-11), 1.92
(2H, m, H-2), 1.88 (1H, m, H-1a), 1.55 (1H, m, H-9), 1.53 (1H,
m, H-7a), 1.52 (1H, m, H-6a), 1.51 (1H, m, H-22a), 1.44 (2H,
m, H-21), 1.39 (1H, m, H-6b), 1.38 (1H, m, H-22b), 1.37 (1H,
m, H-18), 1.36 (1H, m, H-19), 1.35 (1H, m, H-7b), 1.15 (1H,
brs, H-15a), 1.09 (3H, s, H-27), 1.08 (1H, m, H-1b), 1.00 (1H,
m, H-15b), 0.98 (6H, s, H-25, 26), 0.92 (3H, s, H-24), 0.91 (3H,
d, J ) 5.8 Hz, H-30), 0.89 (3H, s, H-23), 0.87 (1H, m, H-20),
0.85 (2H, m, H-16), 0.82 (1H, m, H-5), 0.79 (3H, d, J ) 5.9 Hz,
H-29); 13C NMR (CDCl3, 100 MHz) δ 38.6 (C-1), 23.8 (C-2),
81.1 (C-3), 38.1 (C-4), 55.4 (C-5), 18.3 (C-6), 32.9 (C-7), 40.2
(C-8), 47.7 (C-9), 36.9 (C-10), 23.5 (C-11), 125.1 (C-12), 138.9
(C-13), 42.2 (C-14), 26.1 (C-15), 23.7 (C-16), 38.1 (C-17), 54.2
(C-18), 39.5 × 2 (C-19, 20), 30.7 (C-21), 35.3 (C-22), 28.2 (C-
23), 17.0 (C-24), 15.8 (C-25), 16.8 (C-26), 23.4 (C-27), 69.9 (C-
28), 17.5 (C-29), 21.4 (C-30), 127.2 (C-1′), 114.0 (C-2′), 144.8
(C-3′), 147.2 (C-4′), 115.3 (C-5′), 122.1 (C-6′), 150.0 (C-7′), 115.7
(C-8′), 167.9 (C-9′); HRFABMS m/z 627.4072 [M + Na]+ (calcd
for C39H56O5Na, 627.4025).
Compound 5: yellow amorphous powder; [R]D -31.2° (c 0.5,
MeOH); IR (KBr) νmax 3499, 1710, 1641, 1601, 1515, 1273,
1132, 1073, 1029 cm-1; 1H NMR (CD3OD, 400 MHz) δ 7.67
(1H, dd, J ) 8.4, 1.8 Hz, H-6′′′), 7.53 (1H, d, J ) 1.8 Hz, H-2′′′),
7.00 (1H, d, J ) 8.4 Hz, H-5′′′), 6.89 (1H, d, J ) 8.2 Hz, H-5),
6.64 (1H, d, J ) 8.4 Hz, H-5′), 6.63 (1H, d, J ) 1.8 Hz, H-2),
6.52 (1H, brs, H-2′), 6.50 (1H, m, H-6′), 6.31 (1H, dd, J ) 8.2,
1.7 Hz, H-6), 4.83 (1H, m, H-1′′), 4.65 (1H, dd, J ) 11.7, 2.1
Hz, H-6′′a), 4.44 (1H, dd, J ) 11.7, 7.3 Hz, H-6′′b), 3.89 (3H,
s, 4′′′-OMe), 3.81 (3H, s, 3′′′-OMe), 3.76 (1H, m, H-5′′), 3.73
(3H, s, 3-OMe), 3.68 (3H, s, 3′-OMe), 3.55 (4H, brd, J ) 4.9
Hz, H-9, 9′), 3.52 (1H, m, H-2′′), 3.51 (1H, m, H-3′′), 3.44 (1H,
m, H-4′′), 2.61 (2H, m, H-7a, 7′a), 2.52 (2H, m, H-7b, 7′b), 1.83
(2H, m, H-8, 8′); 13C NMR (CD3OD, 100 MHz) δ 137.3 (C-1),
114.4 (C-2), 150.4 (C-3), 146.0 (C-4), 117.6 (C-5), 122.6 (C-6),
Journal of Natural Products, 2004, Vol. 67, No. 11 1923
36.2 (C-7), 44.1 (C-8), 62.1 (C-9), 133.8 (C-1′), 113.3 (C-2′), 144.8
(C-3′), 145.6 (C-4′), 115.8 (C-5′), 122.7 (C-6′), 36.2 (C-7′), 44.1
(C-8′), 62.1 (C-9′), 102.8 (C-1′′), 74.9 (C-2′′), 77.8 (C-3′′), 72.1
(C-4′′), 75.6 (C-5′′), 65.2 (C-6′′), 123.8 (C-1′′′), 113.7 (C-2′′′),
150.2 (C-3′′′), 155.0 (C-4′′′), 112.0 (C-5′′′), 125.1 (C-6′′′), 167.6
(C-7′′′), 56.6 × 3 (3, 3′′′, 4′′′-OMe), 56.3 (3′-OMe); HRFABMS
m/z 711.2655 [M + Na]+ (calcd for C35H44O14Na, 711.2629).
Compound 6: yellow amorphous powder; [R]D -78.6° (c 0.3,
MeOH); IR (KBr) νmax 3387, 1710, 1659, 1591, 1505, 1461,
1417, 1334, 1228, 1128, 764 cm-1; 1H NMR (CD3OD, 400 MHz)
δ 7.36 (2H, brs, H-2′, 6′), 6.24 (1H, dd, J ) 6.3, 2.1 Hz, H-3),
5.51 (1H, d, J ) 2.5 Hz, H-1), 5.06 (1H, m, H-6), 4.99 (1H, dd,
J ) 6.3, 2.7 Hz, H-4), 4.66 (1H, d, J ) 7.9 Hz, Glc-1), 3.88
(1H, m, Glc-6a), 3.87 (6H, s, H-3′, 5′), 3.82 (3H, s, H-4′), 3.65
(1H, dd, J ) 11.8, 5.8 Hz, Glc-6b), 3.36 (1H, t, J ) 8.7 Hz,
Glc-3), 3.34 (1H, m, Glc-5), 3.26 (1H, m, Glc-4), 3.18 (1H, t, J
) 8.7 Hz, Glc-2), 3.01 (1H, m, H-5), 2.62 (1H, brd, J ) 9.0 Hz,
H-9), 2.28 (1H, dd, J ) 14.3, 6.2 Hz, H-7a), 2.06 (1H, dd, J )
14.3, 3.4 Hz, H-7b), 1.41 (3H, s, H-10); 13C NMR (CD3OD, 100
MHz) δ 93.5 (C-1), 141.3 (C-3), 104.4 (C-4), 39.6 (C-5), 81.2
(C-6), 47.7 (C-7), 79.2 (C-8), 51.8 (C-9), 26.2 (C-10), 126.9 (C-
1′), 108.2 × 2 (C-2′, 6′), 154.4 × 2 (C-3′, 5′), 143.7 (C-5′), 56.8
× 2 (3′, 5′-OMe), 61.2 (3′-OMe), 167.4 (C-7′), 99.4 (Glc-1), 74.8
(Glc-2), 78.0 (Glc-3), 71.7 (Glc-4), 78.3 (Glc-5), 62.9 (Glc-6);
HRFABMS m/z 565.1870 [M + Na]+ (calcd for C25H34O13Na,
565.1897).
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